Quencher-free Molecular Beacon Research Articles

Optimization of Graphene Oxide-based Quencher-free Molecular Beacon for Meat Product Authentication.

Sensitivity is very important in DNA detection. Various attempts have been made to increase detection sensitivity, including increasing the detection capabilities of devices and using DNA probes. This study was aimed to develop a DNA detection method using a quencher-free molecular beacon (QFMB) probe with the help of graphene oxide (GO) as a quencher. The GO has the specific ability to adsorb DNA in the form of a single strand but not in a double strand. The optimum interaction between the MB probe and the target DNA (pig DNA) could produce a double-stranded DNA (dsDNA) so that it is detached from the GO surface. The dsDNA that escapes from the surface of the GO can be detected using a spectrofluorometric technique at an excitation wavelength of 482 nm and an emission of 519 nm, with an intensity comparable to its concentration. The optimum condition that can be used is a GO concentration of 5 μg mL-1, a reaction temperature of 65°C, an incubation time of 6 min, a reaction pH of 7.5 and cation levels of 40 nM. Analysis of the target pork meatball DNA carried out at a concentration interval of 0-500 pg mL-1. So it was concluded that the DNA detection system uses a combination of a quencher-free molecular beacon and graphene oxide, providing a good prospect to be developed into a new method in the halal authentication of meat products using the spectrofluorometric method.

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.

PH-Responsive quencher-free molecular beacon systems containing 2'-deoxyuridine units labeled with fluorene derivatives.

Three fluorescent nucleosides-UFL, UAF, and UDAF, containing fluorene, 2-aminofluorene, and 2-dimethylaminofluorene units, respectively, covalently attached to 2'-deoxyuridine-have been incorporated into the central positions of oligodeoxynucleotides (ODNs) to examine the effects of their flanking bases (FBs) and pH on the emission properties upon hybridization with fully matched and single-base-mismatched targets. The ODN containing UFL and cytosine-FBs in the pH range from 5.5 to 8.0 and the ODN containing UDAF and cytosine-FBs under slightly acidic conditions (pH 6.0-6.5) exhibited dramatic increases in fluorescence only upon duplex formation with their fully matched target DNAs.

Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification.

This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). ExoIII can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by ExoIII. During the digestion of MB2, the 2-aminopurine is separated from the DNA strands and released free in solution, inducing an increase of the fluorescent signal. Owing to the presence of a recessed 3'-terminus in the formed double-stranded DNA, ExoIII-assisted recyclable cleavage of MB2 was achieved. Therefore, an amplified fluorescence signal was observed. Under the optimized conditions, Dam MTase can be detected in the range of 0.2-40units/mL with a limit of detection of 0.2units/mL and good selectivity. Furthermore, the present assay can be used for screening potential DNA MTase inhibitors. Graphical Abstract A quencher-free fluorescence assay for sensitive detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification is reported.

A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid)

A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA in living cells was demonstrated.

Quencher-free molecular beacon tethering 7-hydroxycoumarin detects targets through protonation/deprotonation

In this study, we synthesized a simple but efficient quencher-free molecular beacon tethering 7-hydroxycoumarin on d-threoninol based on its pKa change. The pKa of 7-hydroxycoumarin in a single strand was determined as 8.8, whereas that intercalated in the duplex was over 10. This large pKa shift (more than 1.2) upon hybridization could be attributed to the anionic and hydrophobic microenvironment inside the DNA duplex. Because 7-hydroxycoumarin quenches its fluorescence upon protonation, the emission intensity of the duplex at pH 8.5 was 1/15 that of the single strand. We applied this quenching mechanism to the preparation of a quencher-free molecular beacon by introducing the dye into the middle of the stem part. In the absence of the target, the stem region formed a duplex and fluorescence was quenched. However, when the target was added, the molecular beacon opened and the dye was deprotonated. As a result, the emission intensity of the molecular beacon with the target was 10 times higher than that without the target. Accordingly, a quencher-free molecular beacon utilizing the pKa change was successfully developed.

Intermolecular G-quadruplex-based universal quencher free molecular beacon

A simple and universal quencher-free molecular beacon (MB) with low background fluorescence is developed based on an intermolecular G-quadruplex signaling probe. Unlike previous fluorescent MB strategies, it can function without any fluorophore and quencher modifications on its hairpin sequence.

Quencher-free Linear Beacon Systems with Dual Fluorene-labeled Deoxyuridines

The identification of natural genetic variation in human populations is arguably one of the most important spin-offs of the effort toward the completion of the Human Genome Project. Single nucleotide polymorphisms (SNPs) are the most abundant natural genetic variations in the human genome. To satisfy the demand for molecular diagnostic assays for identifying SNPs, Tyagi and Kramer developed fluorogenic hairpin-shaped DNA probes, called “molecular beacons in 1996. Conventional molecular beacons are single-stranded oligodeoxynucleotides (ODNs) probes that exist in solution as stable stem-and-loop structures in which a fluorophore is covalently linked to one end and a quencher is covalently linked to the other end. The stem keeps the fluorophore and quencher of the molecular beacon in extremely close proximity to each other, when they are not bound to their targets; this causes the fluorescence of the fluorophore to be quenched via the fluorescence resonance energy transfer. By contrast, the conformational change induced by binding to a target nucleic acid causes the fluorophore and the quencher to move away from each other, thereby restoring the fluorescence. Therefore, the presence of the target nucleic acid is characterized by a sharp increase in fluorescence intensity. Recently, we reported that fluorescent hairpin oligonucleotides can function as molecular beacons even without the attachment of additional quencher moieties. Such probes can be called “quencher-free molecular beacons. A quencher-free molecular beacon consists of only a single fluorophore (2-ethynylfluorene-labeled deoxyuridine, U) positioned in the hairpin loop. When this probe undergoes DNA hybridization, it can discriminate between fully matched and single-base-mismatched sequences by exhibiting a strong dA-selective fluorescence. When the fluorene unit is intercalated within a DNA duplex as a result of mismatched base pairing, the fluorescence of the U unit is quenched as a result of a photoinduced charge transfer originating from interactions with neighboring nucleobases. In particular, the quencher-free molecular beacon probe having C-flanking bases (C-FBs) shows more efficient fluorescence ON/OFF behavior than do any other combinations of FBs. The only drawback of this quencher-free molecular beacon system is its poor detection sensitivity due to its relatively high intrinsic emission intensity. To solve this problem, we recently positioned a quencher strand containing a deoxyguanine (dG) nucleobase, functioning as an internal quencher, opposite the U unit to reduce the intrinsic fluorescence on hybridization with a probe. A short strand (five-nucleotide) containing all natural nucleotides and dG as an internal quencher was effective in reducing the intrinsic fluorescence of a linear beacon. In another effort to eliminate this drawback, we employed an ODN containing dual U units at specific distances (Figure 1). We observed that using this dual-labeled linear beacon system resulted in a clear improvement in the detection sensitivity for the perfectly matched target. The modified nucleotide U was synthesized through Sonogashira coupling of a 2-ethynylfluorene unit at the 5position of a 2-deoxyuridine base. Incorporations of the fluorene-labeled deoxyuridine U into the central position of the ODN 1 and of the two U units into the positions with one deoxycytosine spacer unit were effected using standard protocols of automated DNA synthesis (Figure 2). The fluorescence properties of fluorophore-containing DNA are strongly dependent on the electron injection and transfer properties of the FBs. In particular, fluorescent ODNs having C-FBs produce efficient fluorescence ON/OFF systems and have higher sensitivity as probes than do any other combinations of FBs. Therefore, we positioned two deoxy-

Quencher-free molecular beacon: Enhancement of the signal-to-background ratio with graphene oxide

We report the highly improved version of quencher-free molecular beacon (QF-MB) system by using graphene oxide (GO) as an external quencher. This QF-MB/GO system provided a higher S/B ratio (31.0) relative to that (2.2) of the same system in the absence of GO, while retaining a high selectivity for fully matched over single-base-mismatched targets.

Design of an ultimate quencher free molecular beacon containing pyrrolocytidine-guanine base pair

A novel quencher free molecular beacon was designed in which fluorophore-labelled pyrrolocytidine was placed away from the stem terminal. This new type of MB was used for the detection of a target DNA with an excellent efficiency.

Design of an efficient self-quenched molecular beacon for SNPs genotyping

Quencher free molecular beacon with an excellent signal to noise ratio was designed and used for SNPs genotyping.

Quencher-free molecular beacon systems with two pyrene units in the stem region

We appended pyrene units covalently onto adenosine and uridine nucleosides (forming A P and U P units, respectively) and then incorporated them into oligonucleotides such that they were positioned in complementary locations in opposite strands in the middle positions of hairpin stems. Systems 1 ( A P U P ) and 3 ( A P A P ) individually exhibit aromatic stacking between the opposing pyrene units in the stems of their hairpins and display in their spectra the photophysical properties of strongly red-shifted bands; in contrast, the U P U P system 2 exhibits quenching spectra. Systems 1 ( A P U P ) and 3 ( A P A P ) behave as effective molecular beacons (MBs) that change color from green to blue upon duplex formation, whereas 2 ( U P U P ) is an effective MB that changes the intensity of its fluorescence upon forming its perfectly matched duplex.

A highly discriminating quencher-free molecular beacon for probing DNA.

We inserted a fluorene-labeled deoxyuridine derivative, synthesized using Sonogashira coupling, efficiently into the loop region of a DNA hairpin using phosphoramidite chemistry. This molecular beacon, which features no additional fluorescence quencher, discriminates between perfect and one-base-mismatched base pairing by changes in its fluorescence intensity. The discrimination factor is 14.7 for the recognition of a single (A/C) base mismatch.